Bacterial cell transformation
50 ml of subcloning efficiency DH5a TM competent cells (2.5 x 10 8 cfu/mg pUC19) (Life Technologies) are gently thawed on ice in an Eppendorf tube. 1-10ng of plasmid DNA is added to the tube, and mixed by flicking the tube. The sample is then incubated on ice for 30 minutes. The cells are heat shocked at 42°C for 45 seconds and chilled on ice for two minutes. 950 ml of LB broth was added to the cells, and they are incubated for one hour at 37°C. Cells were then plated onto LB-agar with the appropriate antibiotics.
Agrobacterium cell transformation
Competent Agrobacterium cells ( C58C1 pGV101 pMP90) are transformed by electroporation. Firstly 40 ml of cells should be thawed on ice. The binary vector plasmid and a helper plasmid pSOUP, added to the cells, and mixed by flicking, and transferred to a pre-cooled electroporation cuvette (Biorad). An electric pulse is applied across the cuvette (field strength = 2.5kV/cm, Capacitance = 25 m F, resistance = 400 W , pulse-length = 8-12mS). The cells are transferred to 1ml YT broth and incubated at 30°C for 3 Hrs. The sample is centrifuged and the top 900ml of broth is discarded. The pelleted cells are resuspended in the remaining 100 ml of medium, plated onto selective YT medium and incubated for 3 days at 30°C.